IMMUNEPOTENT CRP induces DAMPS release and ROS-dependent autophagosome formation in HeLa and MCF-7 cells.

BACKGROUND: IMMUNEPOTENT CRP (ICRP) can be cytotoxic to cancer cell lines. However, its widespread use in cancer patients has been limited by the absence of conclusive data on the molecular mechanism of its action. Here, we evaluated the mechanism of cell death induced by ICRP in HeLa and MCF-7 cells. METHODS: Cell death, cell cycle, mitochondrial membrane potential and ROS production were evaluated in HeLa and MCF-7 cell lines after ICRP treatment. Caspase-dependence and ROS-dependence were evaluated using QVD.oph and NAC pre-treatment in cell death analysis. DAMPs release, ER stress (eIF2-α phosphorylation) and autophagosome formation were analyzed as well. Additionally, the role of autophagosomes in cell death induced by ICRP was evaluated using SP-1 pre-treatment in cell death in HeLa and MCF-7 cells. RESULTS: ICRP induces cell death, reaching CC50 at 1.25 U/mL and 1.5 U/mL in HeLa and MCF-7 cells, respectively. Loss of mitochondrial membrane potential, ROS production and cell cycle arrest were observed after ICRP CC50 treatment in both cell lines, inducing the same mechanism, a type of cell death independent of caspases, relying on ROS production. Additionally, ICRP-induced cell death involves features of immunogenic cell death such as P-eIF2α and CRT exposure, as well as, ATP and HMGB1 release. Furthermore, ICRP induces ROS-dependent autophagosome formation that acts as a pro-survival mechanism. CONCLUSIONS: ICRP induces a non-apoptotic cell death that requires an oxidative stress to take place, involving mitochondrial damage, ROS-dependent autophagosome formation, ER stress and DAMPs' release. These data indicate that ICRP could work together with classic apoptotic inductors to attack cancer cells from different mechanisms, and that ICRP-induced cell death might activate an immune response against cancer cells.

IMMUNEPOTENT CRP induces cell cycle arrest and caspase-independent regulated cell death in HeLa cells through reactive oxygen species production.

BACKGROUND: Regulated cell death (RCD) is a mechanism by which the cell activates its own machinery to self-destruct. RCD is important for the maintenance of tissue homeostasis and its deregulation is involved in diseases such as cervical cancer. IMMUNEPOTENT CRP (I-CRP) is a dialyzable bovine leukocyte extract that contains transfer factors and acts as an immunomodulator, and can be cytotoxic to cancer cell lines and reduce tumor burden in vivo. Although I-CRP has shown to improve or modulate immune response in inflammation, infectious diseases and cancer, its widespread use has been limited by the absence of conclusive data on the molecular mechanism of its action. METHODS: In this study we analyzed the mechanism by which I-CRP induces cytotoxicity in HeLa cells. We assessed cell viability, cell death, cell cycle, nuclear morphology and DNA integrity, caspase dependence and activity, mitochondrial membrane potential, and reactive oxygen species production. RESULTS: I-CRP diminishes cell viability in HeLa cells through a RCD pathway and induces cell cycle arrest in the G2/M phase. We show that the I-CRP induces caspase activation but cell death induction is independent of caspases, as observed by the use of a pan-caspase inhibitor, which blocked caspase activity but not cell death. Moreover, we show that I-CRP induces DNA alterations, loss of mitochondrial membrane potential, and production of reactive-oxygen species. Finally, pretreatment with N-acetyl-L-cysteine (NAC), a ROS scavenger, prevented both ROS generation and cell death induced by I-CRP. CONCLUSIONS: Our data indicate that I-CRP treatment induced cell cycle arrest in G2/M phase, mitochondrial damage, and ROS-mediated caspase-independent cell death in HeLa cells. This work opens the way to the elucidation of a more detailed cell death pathway that could potentially work in conjunction with caspase-dependent cell death induced by classical chemotherapies.